Cell-permeable (ICP) parkin recombinant protein and use thereof

Abstract

Disclosed are improved cell-permeable Parkin recombinant proteins (iCP-Parkin) which have been developed as a protein-based anti-neurodegenerative agent for efficient BBB-penetration to effectively deliver the recombinant protein into the brain. A Parkin protein, a dopaminergic neuronal cell death inhibitor, has been fused with a newly developed advanced macromolecule transduction domain (aMTD) and preferably with a solubilization domain (SD) to increase the solubility/yield and cell-/tissue-permeability of the recombinant protein. In addition, the aMTD/SD-fused recombinant iCP-Parkin protein has shown BBB-permeability. Both in vitro and in vivo, the iCP-Parkin recombinant protein improved motor skills, a typical phenotype of Parkinson's disease, by increasing dopamine level in the brain by suppressing apoptosis of dopaminergic neuron cells. It also can be applicable as a protein-based anti-neurodegenerative agent to treat Parkinson's disease by protecting dopaminergic neuron cells and regulating the secretion of dopamine.

The invention claimed is: 
     
       1. A recombinant protein, which comprises a Parkin protein and an advanced macromolecule transduction domain (aMTD),
 wherein the aMTD is fused to one end or both ends of the Parkin protein and the aMTD consists of the amino acid sequence of SEQ ID NO: 122. 
 
     
     
       2. The recombinant protein according to  claim 1 , wherein one or more solubilization domain (SD)(s) are further fused to the end(s) of one or more of the Parkin protein and the aMTD. 
     
     
       3. The recombinant protein according to  claim 2 , wherein the recombinant protein is represented by any one of the following structural formulae:
   A-B-C; and 
   A-C-B-C 
 wherein A is the aMTD, B is the Parkin protein, and C is the SD(s). 
 
     
     
       4. The recombinant protein according to  claim 2 , wherein the SD(s) have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 799 and 804. 
     
     
       5. The recombinant protein of  claim 4 , wherein the SD(s) are encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 806 and 811. 
     
     
       6. The recombinant protein according to  claim 1 , wherein the Parkin protein has the amino acid sequence of SEQ ID NO: 814. 
     
     
       7. The recombinant protein according to  claim 6 , wherein the Parkin protein is encoded by the polynucleotide sequence of SEQ ID NO: 815. 
     
     
       8. The recombinant protein according to  claim 1 , wherein the aMTD is encoded by the polynucleotide sequence of SEQ ID NO: 362. 
     
     
       9. The recombinant protein according to  claim 1 , wherein the fusion is formed via a peptide bond or a chemical bond. 
     
     
       10. A pharmaceutical composition comprising the recombinant protein of  claim 1  as an active ingredient; and a pharmaceutically acceptable carrier. 
     
     
       11. A method of treating Parkinson's related diseases in a subject comprising:
 administering to the subject a therapeutically effective amount of the recombinant protein of  claim 1 . 
 
     
     
       12. A polynucleotide encoding a recombinant protein which comprises a Parkin protein and an advanced macromolecule transduction domain (aMTD), wherein the aMTD is fused to one end or both ends of the Parkin protein and the aMTD consists of the amino acid sequence of SEQ ID NO: 122. 
     
     
       13. The polynucleotide according to  claim 12 , wherein the sequence of the polynucleotide is represented by SEQ ID NO: 822. 
     
     
       14. The polynucleotide of  claim 12 , wherein one or more solubilization domain (SD)(s) are further fused to the end(s) of one or more of the Parkin protein and the aMTD, and wherein the recombinant protein is represented by any one of the following structural formulae:
   A-B-C; and 
   A-C-B-C 
 wherein A is the aMTD, B is the Parkin protein, and C is the SD(s). 
 
     
     
       15. The polynucleotide according to  claim 14 , wherein the sequence of the polynucleotide is selected from the group consisting of SEQ ID NOs: 824, 828, and 832. 
     
     
       16. A recombinant expression vector comprising the polynucleotide of  claim 12 . 
     
     
       17. A transformant transformed with the recombinant expression vector of  claim 16 . 
     
     
       18. A preparing method of a recombinant protein comprising:
 culturing the transformant of  claim 17  in a culture medium to produce the recombinant protein; and 
 recovering the recombinant protein expressed by the culturing, 
 wherein the recombinant protein comprises a Parkin protein and an advanced macromolecule transduction domain (aMTD), and 
 wherein the aMTD is fused to one end or both ends of the Parkin protein and the aMTD consists of the amino acid sequence of SEQ ID NO: 122.